The present invention relates to a novel glycoglycerophospholipid originating from Mycoplasma fermentans, an antibody against the glycoglycerophospholipid specifically existing in Mycoplasma fermentans, a method for measuring the glycoglycerophospholipid based on the use of the antibody, and a method for detecting Mycoplasma fermentans. 
The present inventors have already found five species of glycoglycerophospholipids (phosphocholine-containing glycoglycerolipids) from MT-4 cells (human helper T cells infected with HTLV-I (human T lymphotropic retrovirus Type I), Miyoshi et al., Gann., 71, 155-156 (1980)). The present inventors have previously found that one of the five species of glycoglycerophospholipids is 6xe2x80x2-O-phosphocholine-xcex1-glucopyranosyl-(1xe2x80x2-3)-1,2-diacyl-sn-glycerol (Shishitsu-Seikagaku-Kenkyu (Studies on Lipid Biochemistry), Vol. 35, pp. 111-114, 1993).
On the other hand, it has been reported that Macodlasma fermentans is an exacerbation factor of human acquired immunodeficiency syndrome (AIDS) (Lo. S., -C. et al., 1991, Science, 251: 1074-1076; U.S. Pat. No. 5,242,820), or Mycoplasma fermentans is a cause of rheumatism (Williams, M. H. et al., 1970, Lancet ii: 277-280).
Various antibodies against mycoplasmas have been hitherto known, and they have been also used for clinical examination. However, the majority of them are antibodies against Mycoplasma pneumoniae or Mycoplasma genitalium. Monoclonal antibodies against these mycoplasmas have been also prepared. However, it is presumed that such an antibody is an antibody which recognizes a protein of a mycoplasma, or simultaneously recognizes a protein and a lipid of a mycoplasma. Further, any of such antibodies does not exhibit specificity to Mycoplasma fermentans (Japanese Patent Laid-open Nos. 63-298, 63-184064, 63-32496, and 5-304990, and U.S. Pat. Nos. 5,158,870, 4,945,041, and 5,242,820). An antibody, which exhibits specificity to Mycoplasma fementans, is disclosed in U.S. Pat. No. 5,242,820. However, this antibody is obtained by using an entire extract of mycoplasmal cells as an immunogen, and thus the antibody is regarded as an antibody which recognizes a protein. Accordingly, if a mycoplasma contained in a body fluid such as a serum which contains various proteins is detected by using this antibody, the antibody highly possibly makes nonspecific binding. Therefore, it may be impossible to expect a high sensitivity. Further, when an antigen is a protein, it is sufficiently assumed that antigenicity disappears due to mutation in an amino acid sequence of the protein.
As far as the present inventors know, it has not been reported that any mycoplasma has a glycoglycerophospholipid containing phosphocholine. Further, no instance has been known, in which a glycoglycerophospholipid originating from a mycoplasma is used as an immunogen to obtain an antibody which exhibits specificity to the glycoglycerophospholipid. Moreover, it has not been known at all as well that the antibody, which exhibits the specificity to the glycoglycerophospholipid, exhibits high specificity to Mycoplasma fementans. 
It has been reported that the process of pathology of a patient infected with a human immunodeficiency virus is accelerated to arrive at AIDS by infection of a mycoplasma (1991, Science, 251: 4991). It has been also reported that Mycoplasma fementans is an exacerbation factor of AIDS as described above. However, there is no means to correctly detect such behavior of Mycoplasma fermentans in vivo. Therefore, it has been desired to provide an antibody capable of immunologically detecting Mycoplasma fementans in vivo.
The present invention has been made considering a viewpoint as described above, an object of which is to elucidate a glycoglycerophospholipid specifically existing in Mycoplasma fementans, and provide an antibody against the glycoglycerophospholipid, a method for measuring the glycoglycerophospholipid based on the use of the antibody, and a method for detecting Mycoplasma fementans. 
In order to elucidate abnormal proliferation of cells, destruction of cells, and immunological abnormality caused by infection with a retrovirus, the present inventors have analyzed lipids of such infected cells. During this process, the present inventors have confirmed that a glycoglycerophospholipid (phosphocholine-containing glycoglycerolipid) extracted from a human helper T cell strain is unexpectedly a glycoglycerophospholipid originating from Mycoplasma fermentans (phosphocholine-containing glycoglycerolipid: hereinafter simply referred to as xe2x80x9cglycoglycerophospholipid). Further, the present inventors have elucidated a structure of the lipid. Moreover, the present inventors have prepared an antibody against the lipid, and confirmed specificity to various mycoplasmas. As a result, the present inventors have found out that the antibody specifically recognizes Mycoplasma fementans. Thus the present invention has been completed.
Namely, the present invention lies in a glycoglycerophospholipid extractable from Mycoplasma fermentans, having the following properties:
(A) the glycoglycerophospholipid is reactive with orcinol reagent, Dittmer reagent, Dragendorff reagent, and ninhydrin reagent;
(B) the glycoglycerophospholipid is degradable with alkali;
(C) the glycoglycerophospholipid is obtained as a non-adsorptive fraction upon fractionation with an anion exchanger having diethylaminoethyl group; and
(D) the glycoglycerophospholipid has a molecular weight of 1048+28n measured by using a mass spectrometer, wherein n is xe2x88x921, 0, 1, or 2.
The glycoglycerophospholipid described above highly possibly comprises constitutional components of xcex1-glucopyranosyl-(1xe2x80x2-3)-1,2-diacyl-sn-glycerol, phosphocholine, and phosphoric ester of aminopropanediol.
In another aspect, the present invention lies in an anti-glycoglycerophospholipid antibody having reaction specificity to a glycoglycerophospholipid comprising at least phosphocholine, glucose, fatty acid, and glycerol, the lipid being non-adsorptive to an anion exchanger having diethylaminoethyl group, and unstable against alkali. The glycoglycerophospholipid includes, for example, a glycoglycerophospholipid specifically existing in Mycoplasma fementans. 
The present invention provides, as specified embodiments of the anti-glycoglycerophospholipid antibody, an anti-glycoglycerophospholipid polyclonal antibody which reacts with both of 6xe2x80x2-O-phosphocholine -xcex1-glucopyranosyl-(1xe2x80x2-3)-1,2-diacyl-sn-glycerol and the glycoglycerophospholipid extractable from Mycoplasma fermentans, having the following properties, and an anti-glycoglycerophospholipid monoclonal antibody which has reaction specificity to the glycoglycerophospholipid having the following properties:
(A) the glycoglycerophospholipid is reactive with orcinol reagent, Dittmer reagent, Dragendorff reagent, and ninhydrin reagent;
(B) the glycoglycerophospholipid is degradable with alkali;
(C) the glycoglycerophospholipid is obtained as a non-adsorptive fraction upon fractionation with an anion exchanger having diethylaminoethyl group; and
(D) the glycoglycerophospholipid has a molecular weight of 1048+28n measured by using a mass spectrometer, wherein n is xe2x88x921, 0, 1, or 2.
In further aspects, the present invention provides a method for measuring a glycoglycerophospholipid, comprising the step of immunologically measuring the glycoglycerophospholipid having the foregoing properties contained in a specimen, by using the foregoing anti-glycoglycerophospholipid antibody, and a method for detecting Mycoplasma fementans, comprising the steps of measuring a glycoglycerophospholipid contained in a specimen in accordance with the foregoing method, and relating the presence or absence of the glycoglycerophospholipid or an existing amount thereof to the presence or absence of Mycoplasma fementans or an existing amount thereof in the specimen.
In still another aspect, the present invention provides a method for measuring the glycoglycerophospholipid having the foregoing properties (A) to (D) and/or a substance having antigenicity similar to that of the glycoglycerophospholipid contained in a specimen, comprising the step of immunologically measuring the glycoglycerophospholipid and/or the substance having the antigenicity similar to that of the glycoglycerophospholipid by using the foregoing anti-glycoglycerophospholipid antibody.
In still another aspect, the present invention provides a reagent kit for detecting Myconlasma fermentans or a glycoglycerophospholipid of Mycoplasma fermentans contained in a specimen in accordance with an immunological method, comprising the foregoing anti-glycoglycerophospholipid antibody, and a glycoglycerophospholipid of Mycoplasma fementans labeled with a label substance, or a secondary antibody obtained by labeling an antibody against immunoglobulin of an immunized animal with a label substance, the antibody against the immunoglobulin of the immunized animal being prepared by using an animal other than the immunized animal used to prepare the anti-glycoglycerophospholipid antibody.
In still another aspect, the present invention provides a method for detecting a disease selected from AIDS, nephritis, and HTLV-I associated myelopathy, comprising the step of detecting the glycoglycerophospholipid having the foregoing properties (A) to (D), a substance having antigenicity similar to that of the glycoglycerophospholipid, or an antibody having reaction specificity to the glycoglycerophospholipid contained in blood.
In this specification, the glycoglycerophospholipid specifically existing in Mycoplasma fementans is simply referred to as xe2x80x9cglycoglycerophospholipidxe2x80x9d, if necessary.
The present invention will be explained in detail below.
 less than 1 greater than  Glycoglycerophospholipid of the present invention
The antibody of the present invention has reaction specificity to a glycoglycerophospholipid of Mycoplasma fermentans, which is prepared by using the glycoglycerophospholipid as an antigen. At first, the glycoglycerophospholipid of Mycoplasma fementans will be explained.
Existence of lipids inherent in cells infected with a retrovirus has been hitherto known. Five species of glycoglycerophospholipids (GGPLs: GGPL-I, GGPL-II, GGPL -III, GGPL-IV, GGPL-V) have been found in human helper T cells infected with HTLV-I. Among them, the structure of GGPL-I has been determined by the present inventors (Nishida et al., Nippon Nogeikagaku Kaishi, Vol. 68, No. 3 (1994), xe2x80x9cProceedings of 1994th Annual Meetingxe2x80x9d, p. 39). As for GGPLs other than GGPL-I, only their existence is acknowledged, and neither their physical properties nor their structures have been known.
The present inventors prepared lipid fractions from HTLV-I-infected human helper T cells (MT-4 (GGPL+)) in which GGPLs were found, and from cells of MT-4 (GGPLxe2x88x92) obtained by treating the cells of MT-4 (GGPL+) with an anti-mycoplasmal agent (MC201, produced by Dainippon Pharmaceutical). The prepared lipid fractions were separated by HPTLC (high-performance thin layer chromatography). Glycolipid was stained with the orcinol reagent, and phospholipid was stained with Dittmer reagent. As a result, two bands were detected (FIG. 4), which were found in MT-4 (GGPL+), and were not found in MT-4 (GGPLxe2x88x92). The two bands were also detected from MT-4 (GGPLxe2x88x92) cells obtained by cultivation with addition of a culture supernatant of MT-4 (GGPL+) cells previously passed through a filter having a pore size of 0.22 xcexcm. As explained in Examples described later on, it was found that one of the two bands was GGPL-I, and the other was GGPL-III.
It has not been reported that any microorganism belonging to the genus Mycoplasma has, as a constitutional component, a glyceroglycophospholipid containing phosphocholine. It has been considered that GGPLs originate from human cells. However, it has been clarified from the result described above that GGPLs originate from Mycoplasma fementans. Therefore, it is assumed that Mycoplasma fementans can be detected if an antibody having reaction specificity to the glyceroglycophospholipid described above is obtained.
The glycoglycerophospholipid of Mycoplasma fermentans can be prepared from a culture of Mycoplasma fermentans. Mycoplasma fementans can be obtained, for example, as follows. A culture supernatant of cultured cells in which existence of GGPLs is confirmed (xe2x80x9cGGPL -positivexe2x80x9d), for example, a culture supernatant of MT-4 cells (human helper T cells infected with HTLV-I (human T lymphotropic retrovirus Type I)) is filtrated with a filter having a pore size of 0.22 xcexcm. Thus Mycoplasma fermentans is obtained in a filtrate. Alternatively, it is also allowable to use a type strain such as Mycoplasma fementans PG18 strain.
Obtained Mycoplasma fementans is cultivated with, for example, an agar medium of PPLO broth (produced by Difco Laboratories) to isolate a colony which is then cultivated in a liquid medium such as PPLO broth (produced by Difco Laboratories) containing 10% (v/v) fetal bovine serum (FBS), 5% (w/v) yeast extract (produced by Flow Laboratories), 1,000 units/ml penicillin, 1% (w/v) dextrose, and 0.002% (w/v) phenol red. Thus cultivated microbial cells of Mycoplasma fermentans are obtained. Next, methanol is added to the microbial cells,of Mycoplasma fementans, followed by being left to stand for several hours. The thus-obtained microbial cells are then subjected to ultrasonic treatment after chloroform is added thereto. The ultrasonic treated microbial cells is allowed to stand for several hoursxe2x80x94therefor. Subsequently, the microbial cells are homogenized by using, for example, a Potter type TEFLON homogenizer to recover a supernatant. A lipid extract is obtained by evaporating the supernatant. The lipid extract thus obtained contains the glycoglycerophospholipid.
As described in Examples, the lipid fraction was applied to an HPTLC (high-performance thin layer chromatography) plate, which was developed with a mixed solvent of chloroform: methanol: 0.2% (w/v) calcium chloride aqueous solution=50:45:10 (v/v/v) to analyze phospholipid. As a result, six bands (Lipid i, Lipid ii, Lipid iii, Lipid iv, Lipid v, and Lipid vi) were detected (FIG. 3). According to the behavior on TLC, it was revealed that Lipid v and Lipid vi of them corresponded to GGPL-I and GGPL-III respectively. According to a result of FAB mass spectrometry, it was confirmed that GGPL-I was identical with Lipid v, and GGPL-III was identical with Lipid vi.
The glycoglycerophospholipid of Mycoplasma fermentans can be also obtained from MT-4 cells infected with Mycoplasma fementans. For example, MT-4 cells are cultured in RPMI-1640 medium added with 10% (v/v) FCS (fetal calf serum), and obtained cells are washed with PBS (phosphate buffered saline), from which lipids are extracted by using 400 ml of a mixed solution of chloroform: methanol (=2:1, 1:1, or 1:2).
The lipid extract of Mycoplasma fementans or MT-4 cells obtained as described above is divided into a non-adsorptive fraction (neutral fraction) and an adsorptive fraction (acid fraction) by using an anion exchange resin having diethylaminoethyl (DEAE) group such as DEAE-Sephadex A25 (produced by Pharmacia), and thus the non-adsorptive fraction is obtained. The non-adsorptive fraction is applied to a silica bead column, and fractionated with a concentration gradient based on chloroform/methanol/water (83:16:0.5 to 20:80:8 (v/v)). Thus GGPL-III can be separated from other phospholipids. Further, GGPL-I is isolated by performing elution with a concentration gradient based on 1-propanol/aqueous ammonia/water (80:5:15 to 75:5:20).
Both of GGPL-I and GGPL-III are positive to the orcinol reagent, Dittmer reagent, and Dragendorff reagent (which stains choline), and they are degraded by a treatment with mild alkali. GGPL-I is negative to the ninhydrin reaction (which stains amino group), however, GGPL-III is positive to this reaction.
Results of physicochemical analysis performed by using purified GGPL-III are shown below.
(1) Infrared Absorption Spectrum
Absorption bands corresponding to xe2x80x94CH2 and xe2x80x94CH3 groups, hydroxyl group, estercarbonyl group, phosphate group, choline group, and primary amine group were detected respectively.
(2) Liquid Secondary Ionization Mass Spectrometry (LSIMS)
It was suggested that at least three species of fatty acids were present in the molecule. It was concluded that GGPL-III existed as at least four species of molecules, a major component of which had a molecular weight of 1048. Further, it was demonstrated that species of GGPL-III having molecular weights of 1020, 1076, and 1104 were also present.
(3) Tandem Mass Spectrometry (MS/MS)
It was suggested that phosphocholine was present in the molecule. It was concluded that the major component of GGPL-III had a molecular weight of 1048.
(4) Unidimensional 1H NMR Spectrum
Signals of glycerol, choline, and glucose were detected. Further, it was postulated that aminopropanediol was present. Accordingly, the spectrum of GGPL-III was compared with unidimensional 1H NMR spectrums obtained by using standard samples of 3-aminopropane-1,2-diol (1-aminopropane-2,3-diol) and 2-aminopropane-1,3-diol. As a result, it was suggested that GGPL-III possibly contained 2-aminopropane-1,3-diol as a constitutional component.
(5) Two-dimensional 1H NMR Spectrum (PH-DQF-COSY method)
A signal of proton originating from diacylglycerol was observed. A signal of proton of choline was also observed.
(6) Two-dimensional 1H- 31P NMR Spectrum
It was demonstrated that one molecule of GGPL-III contained two phosphorus atoms (P). It was postulated that a compound containing phosphorus bound to 1- or 6-position of a glucose residue. This position was highly possibly 6-position.
A structure of phosphocholine was suggested, in which a phosphate group bound to choline. A structure was postulated, in which a phosphate ester of aminopropanediol firstly bound to the 6-position of the glucose residue, and phosphocholine bound to the phosphoric ester of aminopropanediol.
According to the results described above, it has been revealed that GGPL-III is a novel glycoglycerophospholipid having the following properties:
(A) the glycoglycerophospholipid is reactive with orcinol reagent, Dittmer reagent, Dragendorff reagent, and ninhydrin reagent;
(B) the glycoglycerophospholipid is degradable with alkali;
(C) the glycoglycerophospholipid is obtained as a non-adsorptive fraction upon fractionation with an anion exchanger having DEAE group; and
(D) the glycoglycerophospholipid has a molecular weight of 1048+28n measured by using a mass spectrometer, wherein n is xe2x88x921, 0, 1, or 2.
According to the results described above, it was demonstrated that GGPL-III contained constitutional components of xcex1-glucopyranosyl-(1xe2x80x2-3)-1,2-diacyl-sn -glycerol, phosphocholine, and phosphate ester of aminopropanediol. The phosphate ester of aminopropanediol was a phosphate ester of 2-aminopropane-1,3-diol. It was postulated that its binding site was 6xe2x80x2-position of the glucose reside of xcex1-glucopyranosyl-(1xe2x80x2-3)-1,2-diacyl-sn-glycerol. It was further suggested that phosphocholine bound to the phosphate ester of 2-aminopropane-1,3-diol. The major component of GGPL-III had two palmitoyl groups as acyl groups, having its deduced structure as represented by the following formula (I). It was postulated that the major component of GGPL-III had the structure in which the phosphoric ester of 2-aminopropane-1,3-diol was inserted between phosphocholine and the glucose residue of GGPL-I (6xe2x80x2-O-phosphocholine-xcex1-glucopyranosyl-(1xe2x80x2-3) -1,2-dipalmitoyl-sn-glycerol). 
On the other hand, the other GGPL-III molecules having different molecular weights involve different types of acyl groups in xcex1-glucopyranosyl-(1xe2x80x2-3)-1,2-diacyl-sn-glycerol. It is postulated that the molecule having a molecular weight of 1020 has a myristoyl group and a palmitoyl group, the molecule having a molecular weight of 1076 has a palmitoyl group and a stearoyl group, and the molecule having a molecular weight of 1104 has two stearoyl groups or a palmitoyl group and an eicosanoyl group. However, details are not clarified.
 less than 2 greater than  Preparation of Antibody of the Present Invention
The antibody of the present invention is obtained by immunizing an animal with an antigen of the glycoglycerophospholipid originating from Mycoplasma fermentans, and separating serum from the animal. Alternatively, the antibody of the present invention is obtained by collecting antibody-producing cells of the animal, rendering the antibody-producing cells permanently culturable, and recovering the antibody from a culture thereof. The method for preparing the antibody of the present invention will be exemplarily described below. However, there is no limitation thereto. The antibody of the present invention may be prepared in accordance with other methods provided that the glycoglycerophospholipid of Mycoplasma fementans is used as an antigen.
(1) Preparation of Polyclonal Antibody
Monophosphate lipid, Freund""s complete adjuvant, and mineral oil are added to and mixed with the lipid extract of Mycoplasma fementans obtained as described above. PBS (phosphate buffered saline) containing 0.1% (v/v) Tween 80 is added thereto and emulsified.
Next, an obtained emulsion is subcutaneously or intraperitoneally administered to an animal such as mouse, rat, rabbit, guinea pig, or sheep. After priming immunization, boosting immunization is performed two or three weeks later in accordance with an ordinary method. Thus an antiserum having a high titer is obtained. Blood is collected one week after the final immunization, and serum is separated. The serum is heat-treated to inactivate complement. After that, an immunoglobulin fraction is obtained in accordance with a method similar to those used to purify an ordinary antibody, such as salting out with ammonium sulfate and ion exchange chromatography. Desirably, the increase in antibody titer in blood is confirmed in accordance-with, for example, enzyme immunoassay after the final immunization.
The antibody obtained as described above has reaction specificity to the glycoglycerophospholipid of Mycoplasma fementans, and it does not react with glycoglycerophospholipids of other mycoplasmas such as Mycoplasma arthritidis and Mycoplasma hominis. 
A polyclonal antibody having reaction specificity to GGPL-III can be obtained by using purified GGPL-III instead of the lipid extract of Mycoplasma fementans. 
(2) Preparation of Monoclonal Antibody
A monoclonal antibody is obtained in accordance with a method of Kohler and Milstein (Nature, pp. 495-492, 1975). Namely, antibody-producing cells of a mammalian, which produce an antibody against the glycoglycerophospholipid, is fused with myeloma cells to produce hybridomas. A hybridoma, which produces an objective antibody, is cloned, and the hybridoma is cultured. Thus the monoclonal antibody is obtained in a culture liquid. This process will be explained below while dividing the process into respective steps.
(i) Immunization of Animal and Preparation of Antibody-Producing Cells
Cells, which produce the antibody against the glycoglycerophospholipid, are obtained by immunizing an animal such as mouse, rat, rabbit, guinea pig, or sheep with the glycoglycerophospholipid, and preparing, for example, spleen cells, lymph node cells, or peripheral blood from the animal. The animal may be immunized with the glycoglycerophospholipid in the same manner as described in the item (1).
The monoclonal antibody having reaction specificity to GGPL-III may be obtained by immunizing an animal with purified GGPL-III. Alternatively, the monoclonal antibody may be obtained by preparing hybridomas by using antibody-producing cells of an animal immunized with a glycoglycerophospholipid mixture, and selecting a strain from the obtained hybridomas, the strain producing a monoclonal antibody having reaction specificity to GGPL-III. According to the latter method, it is unnecessary to obtain GGPL-III in an amount required to immunize the animal. It is sufficient to prepare GGPL-III in a minute amount of a degree capable of performing detection by the aid of enzyme immunoassay.
(ii) Preparation of Hybridoma
Antibody-producing cells are collected from the animal immunized with the glycoglycerophospholipid to perform cell fusion with myeloma cells. Various mammalian cell strains can be utilized as the myeloma cells to be used for cell fusion. However, it is preferred to use a cell strain which is the same species as the animal used to prepare the antibody-producing cells. In order to distinguish fused cells from non-fused cells after cell fusion, it is preferred to use a myeloma cell strain which has a marker so that non-fused myeloma cells may not survive, and only hybridoma cells may proliferate. For example, 8-azaguanine-resistant strain is deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT), and its nucleic acid synthesis depends on a de novo synthesis pathway. A fused cell (hybridoma) of such a myeloma cell and a normal antibody-producing cell is proliferative in a medium (HAT medium) containing hypoxanthine, aminopterin, and thymidine because the fused cell can synthesize nucleic acid by using a salvage circuit originating from lymphocyte owing to the presence of thymidine and hypoxanthine even when the de novo synthesis pathway is inhibited by aminopterin. On the contrary, the myeloma cells resistant to 8-azaguanine cannot synthesize nucleic acid, and the cells die because the de novo synthesis pathway is inhibited by aminopterin. Further, the antibody-producing cells as the normal cells cannot be cultured for a long period of time. Therefore, only the hybridoma cells produced by fusion of the antibody-producing cells and the myeloma cells can proliferate in the HAT medium. Accordingly, fused cells can be selected from non-fused cells (Science, Vol. 145, p. 709, 1964). A strain which secretes no inherent immunoglobulin is preferably used as the myeloma cell, in view of the fact that it is easy to obtain an objective antibody from a culture supernatant of an obtained hybridoma.
Cell fusion to obtain the hybridoma is performed, for example, as follows. A spleen is excised from the immunized animal, and it is suspended in RPMI 1640 medium to prepare a cell-floating suspension. The spleen cells are mixed with mouse myeloma cells such as SP2/0 cells (azaguanine-resistant, IgG-non-secretable: ATCC CRL-1581) at the logarithmic growth phase so that the ratio of spleen cells to the myeloma cells is about 10:1 to 1:1. After centrifugation, polyethylene glycol having an average molecular weight of 1,000 to 6,000 is added to a precipitate to give a final concentration of 30 to 50%. Thus the spleen cells and the myeloma cells are fused. Fusion may be performed by applying electric pulse to the cell mixture instead of the addition of polyethylene glycol.
The cells having been subjected to the fusion treatment are cultured with, for example, RPMI 1640 medium containing 10% (v/v) fetal calf serum (FCS), and then the cells are floated in a selective medium such as HAT medium. The cells are dispensed and poured into, for example, wells of a 96-well microtiter plate. Thus the cells are cultured so that only hybridomas are allowed to grow.
(iii) Screening for Hybridoma which Produces Antibody Having Reaction Specificity to Glycoglycerophospholipid
The hybridomas obtained as described above are provided as a mixture of hybridomas which produce monoclonal antibodies against a plurality of antigens or epitopes respectively. Accordingly, it is significant to select, from the hybridomas, a strain which produces a monoclonal antibody having reaction specificity to the glycoglycerophospholipid, especially a monoclonal antibody having reaction specificity to GGPL-III. It is preferred to select a strain which produces a monoclonal antibody against an epitope having strong antigenicity, from monoclonal antibodies which bind to GGPL-III.
A strain, which produces a monoclonal antibody against the glycoglycerophospholipid, can be selected in accordance with an enzyme immunoassay by using the glycoglycerophospholipid as an antigen. Such a method includes an ELISA method comprising the following steps. Namely, the antigen is immobilized on a solid phase such as a microtiter plate, to which a culture liquid of a hybridoma is added, followed by addition of a secondary antibody labeled with, for example, an enzyme, a fluorescent substance, or a luminescent substance to perform incubation . Thus the antibody is detected by the aid of the bound label substance. In this method, an antibody may be immobilized on a solid phase, to which the antigen and a labeled secondary antibody may be successively added, followed by incubation. The ELISA method will be described in detail later on.
When no purified GGPL-III is obtained, the lipid fraction of Mycoplasma fementans is separated by using a high-performance thin layer chromatography (HPTLC) plate. A culture liquid of a hybridoma and a labeled secondary antibody are successively added to the plate, followed by incubation so that a position at which the label substance makes binding is detected. If the position is identical with a position of GGPL-III developed by HPTLC, the hybridoma is regarded to produce a monoclonal antibody against GGPL-III. Once the monoclonal antibody against GGPL-III is obtained, GGPL -III can be also purified from the lipid fraction in accordance with affinity chromatography or the like based on the use of the monoclonal antibody.
When it is confirmed that a well contains a hybridoma which produces an objective monoclonal antibody, cloning is performed from cells in the well containing the hybridoma in accordance with limiting dilution analysis or the like.
As demonstrated in Examples described later on, the hybridoma strain thus obtained, which produces the monoclonal antibody having reaction specificity to GGPL -III, has been deposited on May 24, 1994 in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry under a deposition number of FERM P-14324, transferred to international deposition based on the Budapest Treaty on May 26, 1995, and awarded a deposition number of FERM BP-5115.
(iv) Preparation of Monoclonal Antibody
The monoclonal antibody of the present Invention is obtained in a culture supernatant by culturing the hybridoma obtained as described above in an appropriate medium. The monoclonal antibody can be purified in accordance with an ordinary method including, for example, salting out with ammonium sulfate, ion exchange chromatography, affinity chromatography based on the use of protein A or protein G, and immunoadsorption chromatography based on the use of an immobilized antigen.
The monoclonal antibody thus obtained makes no cross reaction with sialic acid-containing glycolipid (ganglioside) existing in serum of a healthy person not infected with Mycoplasma fementans, platelet activating factor (1-alkyl-2-acetylglycero-3-phosphocholine) or a partially deacylated product thereof, phosphatidylcholine or a partially deacylated product thereof, glycolipid, and phospholipid such as sphingomyelin.
The monoclonal antibody of the present invention can be used as it is. However, those obtained by fragmentation can be also used. Upon fragmentation of the antibody, it is indispensable for binding between the antigen and the antibody that the antigen-binding site (Fab) of the antibody is conserved. Therefore, it is possible to use a fragment containing the antigen-binding site (Fab), obtained by treating the antibody with a protease (for example, plasmin, pepsin, and papain) which does not degrade the antigen-binding site.
If a nucleotide sequence of a gene coding for the monoclonal antibody of the present invention or an amino acid sequence of the antibody is determined, it is possible to produce a fragment containing the antigen-binding site (Fab) in accordance with a technique of genetic engineering.
 less than 3 greater than  Utilization of Glcoglycerphospholipid and Antibody thereagainst of the Present Invention
The antibody of the present invention has reaction specificity to the glycoglycerophospholipid inherent in Mycoplasma fementans. Accordingly, the glycoglycerophospholipid in a specimen can be immunologically measured by using the antibody of the present invention. In this procedure, GGPL-I and GGPL -III can be measured by using the antibody which has reaction specificity to both of GGPL-I and GGPL-III. GGPL-III can be selectively measured by using the antibody which has reaction specificity to GGPL-III. When GGPL-III is measured, GGPL-III obtained as described above can be used as a standard substance. The glycoglycerophospholipid in a specimen includes a lipid fraction extracted from the specimen originating from a living body.
Those applicable to the immunological measurement include ordinary immunological methods based on the use of the antibody, such as ELISA methods and immunostaining methods. For example, the glycoglycerophospholipid in a specimen can be measured by allowing a specimen solution to contact with a solid phase including the anti-glycoglycerophospholipid antibody bound thereto so that the glycoglycerophospholipid contained in the specimen solution is bound to the antibody, separating and removing non-adsorptive components from the solid phase, subsequently allowing a glycoglycerophospholipid originating from Mycoplasma fementans labeled with a label substance to contact with the solid phase, making a competitive reaction between the glycoglycerophospholipid contained in the specimen solution and the labeled glycoglycerophospholipid, and detecting any one of the label substance bound to the solid phase and the label substance not bound to the solid phase.
Alternatively, the glycoglycerophospholipid in a specimen can be measured by allowing a specimen solution and a glycoglycerophospholipid labeled with a standard substance to contact with a solid phase including the anti-glycoglycerophospholipid antibody bound thereto, allowing the glycoglycerophospholipid contained in the specimen solution and the labeled glycoglycerophospholipid to make a competitive reaction with the antibody, and detecting any one of the label substance bound to the solid phase and the label substance not bound to the solid phase. In this procedure, a non-labeled standard glycoglycerophospholipid may be used in place of the labeled glycoglycerophospholipid to perform a competitive reaction between the glycoglycerophospholipid in the specimen and the standard glycoglycerophospholipid, followed by allowing the anti-glycoglycerophospholipid antibody labeled with a label substance to contact with the solid phase so that any one of the label substance bound to the solid phase and the label substance not bound to the solid phase is detected. A labeled secondary antibody may be also used in this procedure.
Further, it is allowable that the glycoglycerophospholipid in a specimen solution is bound to a solid phase, with which a labeled anti-glycoglycerophospholipid antibody is allowed to contact so that any one of the label substance bound to the solid phase and the label substance not bound to the solid phase is detected.
It is also allowable that a standard glycoglycerophospholipid is bound to a solid phase, with which a specimen solution and a labeled anti-glycoglycerophospholipid antibody are allowed to contact so that any one of the label substance bound to the solid phase and the label substance not bound to the solid phase is detected. A labeled secondary antibody may be also used in this procedure. GGPL-III in a specimen can be measured by using purified GGPL-III as the standard glycoglycerophospholipid.
Further, the anti-glycoglycerophospholipid antibody in a specimen can be measured by allowing a specimen solution to contact with a solid phase including the anti-glycoglycerophospholipid bound thereto so that the anti-glycoglycerophospholipid antibody contained in the specimen solution is bound to the antibody, separating and removing non-adsorptive components from the solid phase, subsequently making a reaction with a secondary antibody obtained by labeling an anti-human immunoglobulin antibody with a label substance, and detecting the label substance. The glycoglycerophospholipid of the present invention is inherent in Mycoplasma fementans. Accordingly, it is possible to know the presence or absence of infection with Mycoplasma fementans by inspecting the presence or absence of the anti-glycoglycerophospholipid antibody in a specimen.
Besides the foregoing, various variations are known as methods for the immunological measurement. Any of the methods can be applied to the present invention.
Other than the method based on the use of the solid phase as described above, those adoptable in the present invention include methods to be used for immunologically measuring haptens and antibodies, such as a liquid phase method comprising the steps of allowing a glycoglycerophospholipid in a specimen and a labeled glycoglycerophospholipid to make a competitive reaction with the antibody described above, separating the antigen bound to the antibody from the free antigen by using, for example, polyethylene glycol, dextran, or a secondary antibody, and detecting a label substance of the free labeled antigen.
Those usable as the solid phase include ordinary materials such as agarose beads, latex particles, and wells of microtiter plates composed of, for example, polystyrene or nylon, regardless of their forms (for example, particles, fine particles, test tubes, microtiter plates, and strips). It is preferred to perform blocking by using, for example, BSA (bovine serum albumin) or gelatin after the antibody or the glycoglycerophospholipid is bound to the solid phase. Those usable as the label substance include, for example, enzymes capable of color development of a dye based on an enzyme reaction, such as peroxidase and alkaline phosphatase; radioisotopes; and fluorescent dyes such as fluorescein isothiocyanate.
As for the dye, for example, 4-chloro-1-naphthol, O-phenylenediamine (OPD), or 3,3xe2x80x2-diaminobenzidine is used for peroxidase, and p-nitrophenylphosphate is used for alkaline phosphatase.
Mycoplasma fementans has the glycoglycerophospholipid according to the present invention. Therefore, Mycoplasma fementans can be detected by using the anti-glycoglycerophospholipid antibody of the present invention. For example, Mycoplasma fementans in a specimen can be detected by extracting a lipid fraction from the specimen, allowing the extracted lipid fraction to contact with a solid phase so that the lipid is adsorbed to the solid phase, reacting the solid phase including the lipid adsorbed thereto with the antibody of the present invention, simultaneously or subsequently making a reaction with an anti-immunoglobulin antibody labeled with a label substance and reactive with the antibody, and detecting the label substance.
Further, the glycoglycerophospholipid concerning the present invention is inherent in Mycoplasma fermentans. Therefore, Mycoplasma fementans can be also detected by measuring a glycoglycerophospholipid contained in a specimen in accordance with the method as described above, and relating the presence or absence of the glycoglycerophospholipid or an existing amount thereof to the presence or absence of Mycoplasma fermentans or an existing amount thereof in the specimen.
Those usable as the specimen in the method for detecting the glycoglycerophospholipid or the method for detecting Myconlasma fermentans described above include, for example, blood, serum, plasma, cerebro-spinal fluid, urine, synovial fluid, and cultured cell solution (supernatant).
Besides the foregoing methods, Mycoplasma fermentans can be also detected by reacting the anti-glycoglycerophospholipid antibody labeled with a label substance, with a tissue or cells of a living organism exactly or after applying a treatment for immobilizing a glycoglycerophospholipid, binding the labeled antibody to the tissue or cells of the living organism infected with Mycoplasma fementans, and detecting the label substance. Those usable as a method for the immobilizing treatment include, for example, methods based on the use of formalin, glutaraldehyde, and paraformaldehyde. Alternatively, Mycoplasma fementans may be also detected by reacting a non-labeled anti-glycoglycerophospholipid antibody instead of the anti-glycoglycerophospholipid antibody labeled with the label substance with a tissue or cells of a living organism subjected to an immobilizing treatment, simultaneously or subsequently making a reaction with a secondary antibody obtained by labeling an antibody against immunoglobulin of an immunized animal with a label substance, the antibody against the immunoglobulin of the immunized animal having been prepared by using an animal other than the immunized animal used to prepare the antibody, binding the labeled secondary antibody to the tissue or cells of the living organism infected with Mycoplasma fementans, and detecting the label substance.
Upon detection of the glycoglycerophospholipid or Mycoplasma fementans contained in a specimen by immunological methods, the detection can be conveniently performed by previously preparing a reagent kit comprising the antibody having reaction specificity to the glycoglycerophospholipid of Mycoplasma fementans, and a secondary antibody obtained by labeling an antibody against immunoglobulin of an immunized animal with a label substance, the antibody against the immunoglobulin of the immunized animal being prepared by using an animal other than the immunized animal used to prepare the antibody having reaction specificity to the glycoglycerophospholipid of Mycoplasma fementans. 
The kit is specifically exemplified by a kit comprising, for example, a microtiter plate, a blocking reagent such as BSA (bovine serum albumin), the glycoglycerophospholipid of Mycoplasma fementans (standard substance), the antibody of the present invention, a peroxidase-labeled anti-mouse IgG antibody, an aqueous solution of hydrogen peroxide, OPD, and a washing buffer. It is preferred that the antibodies, the glycoglycerophospholipid of Mycoplasma fementans, and other pertinent components of the kit are provided as lyophilized preparations or dissolved solutions in a solvent capable of stably storing them.
The detection of Mycoplasma fementans can be utilized for the following diagnosis methods.
(1) Prediction of Crisis of Retrovirus Infectious Disease
It is reported that Myconlasma fermentans is an exacerbation factor of AIDS (acquired immunodeficiency syndrome) (Lo, S. -C. et al., 1993, Res. Microbiol., 144, 489-493). Crisis of AIDS does not necessarily occur even when a patient is infected with HIV (human immunodeficiency virus). Usually, AIDS has a long latent period, and complex infection of Mycoplasma fermentans participates in crisis of AIDS. It is conceived that Mycoplasma fementans also participates in crisis of infectious diseases caused by other retroviruses.
Lipids extracted from various species of mycoplasmas were added to a culture liquid of cells latently infected with HIV, at certain lipid concentrations (final concentrations) as shown in Table 1, and the cells were cultured. A number of destroyed cells was inspected by using an optical microscope, which was used as an index of frequency of induction of expression of HIV. Results are shown in Table 1.
According to the results and the foregoing report, it is understood that Mycoplasma fementans has an activity to induce proliferation of the retrovirus. Therefore, it is possible to predict the crisis by inspecting the presence or absence of complex infection of Mycoplasma fementans with, for example, blood of a patient infected with the retrovirus. Thus it is possible to take an appropriate treatment.
(2) DiaQnosis of Rheumatism
It is reported that Mycoplasma fementans is a cause of rheumatism (Williams, M. H. et al., Lancet ii: 277-280 (1970)). Therefore, it is expected that morbidity of rheumatism can be diagnosed by detecting Mycoplasma fementans by using the antibody of the present invention.
(3) Application to Targeting Therapy
It is expected to apply and utilize the antibody of the present invention for preventing crisis of retrovirus diseases and curing rheumatism such that an anti-HIV agent such as azidothymidine or dideoxyinosine and/or an anti-mycoplasmal agent is bound to the antibody of the present invention to be administered to a patient infected with the retrovirus or a patient of rheumatism.
(4) Utilization an Neutralizing Antibody for Treatment
The antibody of the present invention is expected to present an activity as a neutralizing antibody which binds to Mycoplasma fementans to avoid infection caused by Mycoplasma fementans. Administration of the antibody to a living body makes it possible to use the antibody for curing or preventing infectious diseases caused by viruses having high possibility of complex infection of Mycoplasma fementans. 
(5) Diagnosis of Nephritis
As demonstrated in Examples described later on, a lipid, to which the anti-glycoglycerophospholipid antibody of the present invention binds, is highly frequently found in blood of patients of nephritis. It has been suggested that nephritis can be diagnosed by detecting the lipid. The lipid is distinguished from GGPL-III judging from the position on HPTLC. However, the lipid is considered to be a lipid having antigenicity similar to that of GGPL-III because the monoclonal antibody which recognizes GGPL-III binds to the lipid. The substance having antigenicity similar to that of GGPL-III can be immunologically measured by using the anti-glycoglycerophospholipid antibody of the present invention.